help with protein size - western blot (Sep/21/2004 )
Hope someone has a suggestion here. I purchased an antibody from a company whose technical support said that the specificity of the antibody was confirmed by running a Western. Their band showed up around 105-105 kDa. I ran a Western with the antibody (both rat tissue which they used and my test tissue) and I am getting bands for both sets of tissue at about 50-52 kDa. I have tried using protease inhibitors, both an recipe and a inhibitor cocktail, and still have similar results. The antibody is a polyclonal, but I'm not sure how likely it is that it is now recognizing a different protein since it has already been tested in rat with a single band at 105. Also, FYI the protein is a dimer, but literature suggests that each subunit should be about 100-110 kDa, not the dimer. Any suggestions or explanations as to why this protein would be half the size of the normal?
Could you be seeing the heavy chain of the IgG? Have you tried a longer exposure to see if something is present in the 105 range? Try with and w/o BME or boiling.